Our laboratory has been instrumental in demonstrating the abundant presence of inflammatory markers in Alzheimer's disease (AD) brain samples. These data have been confirmed and extended by many independent laboratories. However, because previous studies have emphasized the demonstration of markers rather than the elucidation of mechanisms, doubts have remained concerning whether AD inflammatory processes arc merely a 'sophisticated means of clearing the detritus of already existent AD pathology, or are a significant contributor to that pathology. We now propose to study specific mechanisms of inflammatory processes in the AD brain and their interactions with classic elements of AD pathology such as B-amyloid peptide (Abeta) deposition. Our intent is to span a range of mechanisms from in vitro binding kinetics to culture models of pathophysiology. By their nature, we believe that the mechanisms and interactions elucidated by the proposed work will point to a role for AD inflammation that is not etiologic, but nonetheless contributes substantially to the overall pathogenic process. I. Our preliminary studies show that Abeta binds C1q in an antibody independent fashion, and that this binding leads to full activation of the classical complement cascade. These data have now been replicated by at least three independent laboratories. In vitro experiments with Abeta peptides, variants, and fragments will therefore define equilibrium binding, steric, and macromolecular properties of Abeta/C1q binding and classical pathway activation in the AD brain using in vitro ligand binding (ELISA and radiolabel) methods and complement activation (CH50 and ELISA) assays. Among other variables we will test for pathophysiologic relevance by showing that rat Abeta does not bind or activate C1q, whereas familial Dutch variant Abeta does. 2. Our preliminary studies suggest that C1q enhances formation and stabilizes beta-pleated Abeta aggregates, the form associated with compacted Abeta deposits and neurodegeneration. In vitro experiments using fluorimetry, electron microscopy, and Western blots will confirm and extend these findings. 3. Our preliminary experiments demonstrate that in the presence of complement previously nontoxic concentrations of Abeta (or complement) result in significant morphologic damage to neurites and as much as a five-fold increase in lactic acid dehydrogenase release in embryonic rat brain cultures. Culture experiments will therefore use Abeta peptides, variants, and fragments to elucidate cytopathic mechanisms of interaction between complement and Abeta. 4. In situ experiments with cultured rat embryonic cells and brain tissue from AD and control patients will demonstrate pathophysiologically relevant co-localizations of complement proteins with Abeta and tissue. Our preliminary immunoelectron and light microscopic data show that C1q co-localizes with aggregated Abeta deposits, whereas C5b-9, the membrane attack complex, co-localizes with adjacent neuritic membranes. Notably, we do not find Ig at these sites (despite being able to demonstrate Ig peripherally by the same techniques). We propose to confirm these results in additional AD brain samples and to extend them to the level of mechanism in rat brain cultures where the dynamics of complement activation and lysis can be manipulated experimentally.